Microbial agent for controlling discharge of H2S and CH4 in sewage pipe network as well as preparation method and application of microbial agent

Abstract

The invention relates to the technical field of water treatment, in particular to a microbial agent for controlling discharge of H2S and CH4 in a sewage pipe network as well as a preparation method and application of the microbial agent. The fungicide comprises one or two of bacillus subtilis and saccharomyces cerevisiae. The microbial agent provided by the invention has an efficient inhibition effect on H2S and CH4 in an anaerobic sewage system, the inhibition rates can reach (73 + / -15)% and (85 + / -15)%, and pollution reduction, carbon reduction and pipe network corrosion reduction are facilitated. The carbon flow direction is changed by adding the microbial agent, so that organic acid in the sewage is mainly fatty acid with a long carbon chain, the generation of H2S and CH4 is reduced, and the microbial agent is used as a carbon source to improve the sewage treatment efficiency and the effluent quality. The microbial agent disclosed by the invention shows long-term effectiveness in the aspect of inhibiting generation of H2S and CH4 in an anaerobic sewage system, and the inhibition effect is still stable after continuous operation for 16 days.

Description

Technical Field

[0001] The present invention relates to the technical field of water treatment, and in particular to a method for controlling H 2 S and CH 4 Discharged bacterial agent and its preparation method and application. Background Art

[0002] Due to the closed space and oxygen consumption of organic matter, the sewage in the pipeline is in an anaerobic state. The sulfate-reducing bacteria (SRB) in the pipeline reduce sulfate to sulfide and further produce H 2 S. Gas phase H in sewage pipes 2 The S concentration can reach more than 500 ppm, while the H 2 S is toxic and can kill people at concentrations above 400 ppm. 2 S can also cause odor pollution and pipeline corrosion in residential communities. On the other hand, organic matter is converted into small molecular organic matter, acetic acid and hydrogen under the action of a series of bacterial communities, and further produces CH under the action of methanogens (MA). 4 and CO 2 , CH in the gas phase of the pipeline network 4 The concentration will exceed 5%. 4 It is a greenhouse gas, and its greenhouse effect is 23-25 ​​times that of carbon dioxide. 4 The explosion limit is less than 5%, and CH 4 In summary, H generated and discharged in the sewage pipe network 2 S and CH 4 It is very harmful to production, life and health, causing huge economic losses every year. Therefore, it is necessary to develop efficient H 2 S and CH 4 Collaborative control method.

[0003] Currently, its H 2 S and CH 4 Control methods include chemical and biological methods. Among them, chemical methods include aeration oxygen supply and addition of oxidants, alkali, nitrates, iron salts, free nitrite, free ammonia and other chemical agents. However, chemical methods are costly, have short-lasting effects, and some agents cause large secondary pollution, and may increase the treatment burden of downstream sewage treatment plants. Using microorganisms to inhibit H 2 S and CH 4 Generation is a method with potential application prospects, which is environmentally friendly and sustainable, but the strains suitable for application in sewage pipe networks and their effects still need further exploration. Summary of the invention

[0004] The present invention provides a method for controlling H in a sewage pipe network.2 S and CH 4 The discharged bacterial agent and its preparation method and application are used to solve the above-mentioned problems existing in the prior art.

[0005] According to a first aspect of the present invention, the present invention provides a method for controlling H in a sewage pipe network. 2 S and CH 4 The discharged bacterial agents include one or both of Bacillus subtilis and Saccharomyces cerevisiae.

[0006] The present invention obtains the ability to effectively inhibit H from the source by screening. 2 S and CH 4 The microbial strains produced by Bacillus subtilis and Saccharomyces cerevisiae can effectively inhibit H in sewage pipe networks when used alone or in combination. 2 S and CH 4 The study found that after adding the bacterial agent of the present invention, the accumulation of volatile fatty acids (VFAs) such as propionic acid, butyric acid, and valeric acid in the sewage was significantly improved. The mechanism is that the direction of carbon flow is changed. After adding the bacterial agent, the organic acids in the sewage are mainly propionic acid, butyric acid, and valeric acid. These fatty acids with longer carbon chains need to be further decomposed and fermented before they can be used by sulfate-reducing bacteria and methanogens. This process is slow, resulting in a reduction in available electron donors, thereby inhibiting H 2 S and CH 4 These fatty acids, as biodegradable organic carbon sources, can effectively solve the problem of insufficient carbon sources in the sewage treatment plant during the treatment process, thereby improving the treatment efficiency and effluent quality. This shows that the addition of bacterial agents not only inhibits the emission of harmful gases in the sewage network, but also provides better conditions for the subsequent biological treatment stage. The experiment found that the bacterial agent of the present invention can effectively inhibit the H 2 S and CH 4 In addition, it not only reduces the emission of odorous gases and greenhouse gases, but also delays the 2 S and CH 4 The time to reach the peak concentration means that the generation rate of these harmful gases has been effectively controlled, which is helpful to reduce pollution and carbon emissions and alleviate pipe network corrosion. The bacterial agent selected in the present invention has been verified by multiple water exchange experiments to inhibit H in the anaerobic sewage system. 2 S and CH 4The generation showed long-term effectiveness, and the inhibitory effect was still stable after 16 days of continuous operation. Even under the condition of continuous operation, the inhibitory effect remained stable. This shows that the frequency of replenishing the microbial agent is low, which reduces the cost. In addition, the two selected strains are both commonly used strains that can be mass-produced. Not only do they have mature production processes in industrial production, but they also have high biosafety and will not cause secondary pollution to the environment. Using these strains as microbial agents can not only ensure the feasibility of large-scale application, but also do not require large facilities, are easy to operate, meet environmental protection requirements, and are environmentally friendly.

[0007] In order to achieve better suppression of H 2 S and CH 4 Furthermore, the bacterial agent is prepared by mixing Bacillus subtilis liquid and Saccharomyces cerevisiae liquid in a volume ratio of (1-2): (1-3).

[0008] Optionally, the volume ratio of the Bacillus subtilis solution to the Saccharomyces cerevisiae solution in the bacterial agent can be 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1.5:1, 1.5:2, 2:1, 2:1.5, 2:2.5 or 2:3, etc., preferably 1:(1.5-2.5). Under this condition, the bacterial agent of the present invention has an effect on the H 2 S and CH 4 The inhibition rates can reach (73±15)% and (85±15)%.

[0009] In order to achieve better suppression of H 2 S and CH 4 Further, in the Bacillus subtilis liquid, the effective viable bacteria count of the Bacillus subtilis is (1-2)×10 8 CFU / mL.

[0010] In order to achieve better suppression of H 2 S and CH 4 Furthermore, in the brewer's yeast liquid, the effective viable count of the brewer's yeast is (1-2)×10 8 CFU / mL.

[0011] In a specific embodiment, the sewage indicators in the sewage network are as follows: COD concentration is 321 ± 10 mg / L, VFA concentration is 0.2 ± 0.05 mmol / L, NH 4 + -N concentration was 22.4 ± 1.1 mg N / L, S 2- The concentration was 0.008 ± 0.002 mg S / L, SO 4 2-The concentration was 145.6 ± 22 mg S / L, the DO concentration was 0.15 ± 0.05 mg DO / L, the ORP concentration was -315 ± 15 mV, and the pH was 7.2 ± 0.5.

[0012] According to a second aspect of the present invention, the present invention also provides the above-mentioned control of H in the sewage pipe network 2 S and CH 4 The preparation method of the discharged microbial agent comprises the following steps: The Bacillus subtilis was cultured in LB liquid medium, and then resuspended in a sterile phosphate buffer solution to obtain a Bacillus subtilis bacterial liquid; And / or, culturing the cerevisiae yeast in a YPD liquid medium, and then resuspending in a sterile phosphate buffer solution to obtain a cerevisiae yeast liquid.

[0013] The culture medium is the basis for the growth and reproduction of microorganisms. It provides the necessary nutrition and environmental conditions for the microorganisms. The present invention selects a suitable type of culture medium to culture the strains, and then selects a suitable type of buffer solution for dilution, so as to obtain a Bacillus subtilis liquid and a Saccharomyces cerevisiae liquid with better performance, thereby facilitating the control of H in the sewage pipe network. 2 S and CH 4 emission.

[0014] Furthermore, the method further comprises the following steps: compounding the obtained Bacillus subtilis liquid and the obtained Saccharomyces cerevisiae liquid to obtain the bacterial agent. Furthermore, the LB liquid culture medium uses water as a solvent, and includes 5-15 g / L of peptone, 3-8 g / L of yeast powder, and 5-15 g / L of sodium chloride; the pH value of the LB liquid culture medium is 7.0-7.5, and is sterilized at high temperature at 120-125°C for 10-30 min.

[0015] Preferably, the LB liquid culture medium uses water as a solvent, including 8-12 g / L of peptone, 4-6 g / L of yeast powder, and 8-12 g / L of sodium chloride; the pH value of the LB liquid culture medium is 7.1-7.3, and is sterilized at 120-123°C for 15-25 min. In some specific embodiments, the LB liquid culture medium uses distilled water as a solvent, including 10 g / L of peptone, 5 g / L of yeast powder, and 10 g / L of sodium chloride; the pH value of the LB liquid culture medium is 7.2, and is sterilized at 121°C for 20 min.

[0016] The YPD liquid medium uses water as a solvent, including 15-25 g / L of peptone, 8-12 g / L of yeast extract powder, and 15-25 g / L of glucose; the pH value of the YPD liquid medium is 6.3-6.7, and it is sterilized at 120-123°C for 15-25 min. In some specific embodiments, the YPD liquid medium uses water as a solvent, including 20 g / L of peptone, 10 g / L of yeast extract powder, and 20 g / L of glucose; the pH value of the YPD liquid medium is 6.5, and it is sterilized at 121°C for 20 min.

[0017] Further, the concentration of the sterile phosphate buffer solution is 0.005-0.015 mol / L. Preferably, the concentration of the sterile phosphate buffer solution is 0.008-0.012 mol / L. In some specific embodiments, the concentration of the sterile phosphate buffer solution is 0.01 mol / L.

[0018] Further, Bacillus subtilis was cultured in LB liquid medium at 100-200 r / min at 35-40°C for 12-24 h, and then centrifuged at 4000-6000 rpm for 5-15 minutes to remove the medium, and then diluted to OD 0. 600 is 0.2-0.4, and Bacillus subtilis liquid is obtained.

[0019] Saccharomyces cerevisiae was cultured in YPD liquid medium at 100-200 r / min at 35-40°C for 12-24 h, then centrifuged at 4000-6000 rpm for 5-15 min to remove the medium, and then diluted to OD 0. 600 is 1-1.5 to obtain brewer's yeast liquid.

[0020] According to a third aspect of the present invention, the present invention also provides the above-mentioned control of H in the sewage pipe network 2 S and CH 4 The discharged bacterial agent controls H in the sewage pipe network 2 S and CH 4 Application of emission methods.

[0021] Furthermore, the bacterial agent is directly added to the sewage for use, and the inoculation amount of the bacterial agent is (1-2)×10 7 CFU / mL; or, the bacterial agent is centrifuged to make its water content 50%-70%, and then prepared into a solid bacterial agent and added to the sewage for use; preferably, the dosage of the solid bacterial agent is 0.4-0.5kg / m 3 .

[0022] Beneficial effects of the present invention: The present invention provides a method for controlling H in a sewage pipe network 2 S and CH 4 Effect of discharged bacterial agents on H in anaerobic sewage system 2 S and CH 4 It has a highly effective inhibitory effect, with an inhibition rate of (73±15)% and (85±15)%. In addition, it not only reduces the emission of odorous gases and greenhouse gases, but also delays the release of H 2 S and CH 4 The time to reach the peak concentration. This means that the generation rate of these harmful gases has been effectively controlled, which is helpful to reduce pollution and carbon emissions and alleviate pipe network corrosion. The bacterial agent selected in the present invention has an effective effect on inhibiting H 2 S and CH 4 The generation shows long-term effectiveness. Even under conditions of continuous operation, the inhibitory effect remains stable. This shows that the frequency of supplementing the bacterial agent is low, which reduces costs. The strains of the present invention are all commonly used strains that can be mass-produced. Not only do they have mature production processes in industrial production, but they also have high biosafety and will not cause secondary pollution to the environment. The bacterial agent of the present invention can significantly increase the accumulation of fatty acids such as propionic acid, butyric acid, and valeric acid in sewage. These fatty acids, as biodegradable organic carbon sources, can effectively solve the problem of insufficient carbon source in the treatment process of sewage treatment plants, thereby improving treatment efficiency and effluent water quality. This shows that the addition of bacterial agents can not only inhibit the emission of harmful gases in the sewage network, but also provide better conditions for the subsequent biological treatment stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 The present invention provides a method for controlling H in a sewage pipe network. 2 S and CH 4 In the preparation method of the bacterial agent discharged, the bacterial agent of Example 1-2 and Comparative Example 1-2 has an effect on H 2 S(a) and CH 4 Long-term effects of concentration (b).

[0025] Figure 2 The present invention provides a method for controlling H in a sewage pipe network. 2 S and CH 4 In the preparation method of the bacterial agent discharged, the bacterial agent of Example 1-2 and Comparative Example 1-2 has an effect on H 2S(a) and CH 4 Long-term suppression effect of emissions (b).

[0026] Figure 3 The present invention provides a method for controlling H in a sewage pipe network. 2 S and CH 4 In the method for preparing the bacterial agent discharged, Examples 1-5 treat H 2 S(a) and CH 4 Effect of concentration (b) and inhibitory effect (c).

[0027] Figure 4 The present invention provides a method for controlling H in a sewage pipe network. 2 S and CH 4 Effects of Examples 1, 2, and 5 on the concentrations of total organic carbon (TOC) (a) and VFAs (b) in wastewater in the preparation method of the discharged bacterial agent.

[0028] Figure 5 The present invention provides a method for controlling H in a sewage pipe network. 2 S and CH 4 In the preparation method of the bacterial agent discharged, the application example is to the sewage pipe network H 2 The influence of S concentration. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The components used in the present invention are all purchased from the market: freeze-dried powders of Bacillus subtilis (ACCC 19743) and Saccharomyces cerevisiae (ACCC 21182) are purchased from the China Agricultural Microbiological Culture Collection Administration Center, freeze-dried powder of Lactobacillus acidophilus (CGMCC 1.1878) is purchased from the China General Microbiological Culture Collection Administration Center, and Candida tropicalis (CICC 31592) is purchased from the China Industrial Microbiological Culture Collection Administration Center.

[0031] Example 1 This embodiment provides a method for controlling H in a sewage pipe network. 2 S and CH 4 The discharged bacterial agent is a Bacillus subtilis bacterial solution, and its preparation method is as follows: After activation, Bacillus subtilis was inoculated into LB liquid medium and cultured at 150 r / min at 37°C for 12 h. The culture medium was then removed by centrifugation at 5000 rpm for 10 min. The pure culture of Bacillus subtilis was diluted to OD 0. 600 is 0.3, and the bacterial solution concentration is about 10 8 CFU / mL, and the prepared bacterial solution was used for subsequent experiments.

[0032] The LB medium includes 10 g / L peptone, 5 g / L yeast powder, 10 g / L sodium chloride, and the rest is distilled water. The pH value of the solution is adjusted to 7.2 with HCl, and the solution is sterilized at high temperature at 121° C. for 20 min.

[0033] Example 2 This embodiment provides a method for controlling H in a sewage pipe network. 2 S and CH 4 The discharged bacterial agent is a brewer's yeast liquid, and its preparation method is as follows: After activation, Saccharomyces cerevisiae was inoculated into YPD liquid medium and cultured at 150 rpm for 12 h at 37 °C. The medium was then removed by centrifugation at 5000 rpm for 10 min. The pure culture of Bacillus subtilis was diluted to OD 0.01 with sterile PBS solution. 600 is 1.1, and the bacterial concentration is about 10 8 CFU / mL, and the prepared bacterial solution was used for subsequent experiments.

[0034] The YPD medium includes 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose, and the rest is distilled water. The pH value of the solution is adjusted to 6.5 with HCl, and sterilized at 121° C. for 20 min.

[0035] Comparative Example 1 This comparative example provides a method for controlling H in a sewage pipe network. 2 S and CH 4 The discharged bacterial agent is a Lactobacillus acidophilus bacterial liquid, and its preparation method is as follows: After activation, Lactobacillus acidophilus was inoculated into LB liquid medium and cultured at 150 r / min at 37°C for 48 h. The medium was then removed by centrifugation at 5000 rpm for 10 min. The pure culture of Bacillus subtilis was diluted to OD 0. 600 is 0.3, and the bacterial solution concentration is about 10 8 CFU / mL, and the prepared bacterial solution was used for subsequent experiments.

[0036] The LB medium includes 10 g / L peptone, 5 g / L yeast powder, 10 g / L sodium chloride, and the rest is distilled water. The pH value of the solution is adjusted to 7.2 with HCl, and the solution is sterilized at high temperature at 121° C. for 20 min.

[0037] Comparative Example 2 This comparative example provides a method for controlling H in a sewage pipe network. 2 S and CH 4 The discharged bacterial agent is tropical Candida liquid, and its preparation method is as follows: After activation, Candida tropicalis was inoculated into YPD liquid medium and cultured at 150 r / min at 37°C for 12 h. The medium was then removed by centrifugation at 5000 rpm for 10 min. The pure culture of Bacillus subtilis was diluted to OD 0. 600 is 1.1, and the bacterial concentration is about 10 8 CFU / mL, and the prepared bacterial solution was used for subsequent experiments.

[0038] The YPD medium includes 20 g / L peptone, 10 g / L yeast extract, 20 g / L glucose, and the rest is distilled water. The pH value of the solution is adjusted to 6.5 with HCl, and sterilized at 121° C. for 20 min.

[0039] In the above examples 1-2 and comparative examples 1-2, the obtained liquid bacterial agent can be used directly, and the inoculation amount is 10 7 CFU / mL; it is also centrifuged to make its water content 50%-70% to prepare solid bacterial agent, and the dosage of solid bacterial agent can be controlled at 0.4-0.5 kg / m 3 .

[0040] Test Example 1 The effects of different bacterial formulations on H production in sewage anaerobic system were studied by shake flask experiment. 2 S and CH 4 The synergistic inhibition effect is achieved by shaking the bottle to simulate the flow of sewage in the drainage pipe. The gas sampling bag is connected as the headspace, the thread of the bottle mouth is sealed with raw tape to prevent leakage, and a tetrafluoroethylene gasket is used to reduce the error caused by gas adsorption.

[0041] In order to restore the real drainage pipe environment, sludge was taken from the in-service sewage pipe of Tsinghua University (see Table 1 for specific indicators), located in the green belt beside the road on the north side of the Sino-Italian Environmental Energy Saving Building of Tsinghua University.

[0042] Table 1 Sludge properties

[0043] 25 mL of sewage pipe sludge and 200 mL of artificial domestic sewage were added to the shake flask. Nitrogen was blown into the water to remove oxygen before adding it to the shake flask, so that the DO concentration was lower than 0.2 mg / L, which was consistent with the characteristics of actual domestic sewage. The specific water quality indicators are shown in Table 2.

[0044] Table 2 Artificial sewage water quality indicators

[0045] The blank group was added with 25 mL of PBS solution, and the experimental groups were added with 25 mL of bacterial solution obtained in Examples 1 and 2 (inoculation concentration 10 7 CFU / mL). The flask was sealed and placed in a constant temperature shaker at 25°C and 120 rpm for subsequent experiments. The H in the gas sampling bag was tested every 12 hours. 2 S and CH 4 The concentration changes were measured with a cycle of 96 hours. The sewage in the shake flask was replaced after each cycle. In order to avoid disturbing the sediment at the bottom of the shake flask, the water was allowed to stand for 12 hours before each water change. 100 mL of sewage was slowly sucked out and injected using a syringe. After four cycles, the effect of H 2 S and CH 4 Concentration inhibition effect and effective duration.

[0046] like Figure 1 In the experimental group with Bacillus subtilis culture solution added, H 2 The concentration of S also showed a trend similar to that of the blank group, reaching a peak of 310.75 mg / m at the 60th hour. 3 Then it gradually decreases, and H is produced in the first cycle 2 The average concentration of S was 136.44 mg / m 3 , which was significantly lower than that of the blank group. 2 The concentration of S reached a peak of 616.36 mg / m at 72 h. 3 Compared with the blank group, the peak time was delayed, and H production in the first cycle 2 The average concentration of S was 230.89 mg / m 3 , slightly lower than the blank group. The experimental group treated with tropical Candida showed a fluctuating upward trend in the first cycle and reached a peak of 548.79 mg / m at 84 h. 3 , which was lower than the peak concentration of the blank group, but the H production in the first cycle 2 The average concentration of S was 298.88 mg / m 3 , slightly higher than the blank group. The experimental group with addition of brewer's yeast reached a peak of 197.06 mg / m at 84 h. 3Compared with the blank group, the peak time was delayed and the peak concentration was significantly lower than that of the blank group. 2 The average concentration of S was 79.41 mg / m 3 , which had a significant inhibitory effect compared with the blank group.

[0047] The production of H in the blank group was significantly higher in the second cycle (108-192 h), the third cycle (204-288 h), and the fourth cycle (300-384 h). 2 The average concentration of S was 505.96 mg / m 3 , 593.91 mg / m 3 and 387.13 mg / m 3 The experimental group with Bacillus subtilis produced 2 The average concentration of S was 312.01 mg / m 3 、300.02 mg / m 3 and 266.07 mg / m 3 , were significantly lower than those in the blank group, and the time to reach the peak was delayed by 24 h. 2 The average concentrations of S were lower than those of the blank group and were 361.03 mg / m 3 , 284.35 mg / m 3 and 343.01 mg / m 3 , and the peak time was delayed by more than 24 hours. 2 The average concentration of S was 366.21 mg / m 3 , 344.34 mg / m 3 and 349.27 mg / m 3 , slightly lower than the blank group. 2 The average concentration of S was 133.15 mg / m 3 , 199.42 mg / m 3 and 143.39 mg / m 3 , which was significantly lower than that of the blank group, and the time to reach the peak also showed a trend of being delayed. This experimental result shows that all four strains can inhibit SRB and thus reduce H 2 The effect produced by S.

[0048] like Figure 2 As shown in Figure 2, Saccharomyces cerevisiae inhibits H production in the anaerobic system of sewage. 2S had the best effect, with the highest inhibition rate reaching 73.68% and the average inhibition rate being 68.72%. It showed a relatively stable and superior inhibition effect in all four cycles, but the inhibition rate began to decline slowly from the third cycle. 2 The inhibition rate of S showed a fluctuating downward trend within 4 cycles, with the highest inhibition rate reaching 51.6% and the average inhibition rate being 42.67%. 2 The inhibition rate of S showed a trend of first increasing and then decreasing, reaching a maximum of 52.12% in the third cycle, and an average inhibition rate of 27.56%. The tropical Candida showed a similar trend, with an average inhibition rate of 27.56% in the first cycle. 2 The inhibition rate of S was -6.01%, but it increased to 42.02% in the third cycle, and the average inhibition rate was 18.35%.

[0049] like Figure 1 As shown in Figure 2, in the first cycle (0-96 h), the blank group produced CH 4 The average concentration was 51347.36 mg / m 3 , reaching a peak of 129063.02 mg / m after 72 h of fermentation. 3 Then it began to decrease and dropped to 19932.95 mg / m at 96 hours. 3 In the experimental group with Bacillus subtilis culture solution, CH 4 The concentration reached a peak of 20178.72 mg / m at 72 h. 3 , CH is produced in the first cycle 4 The average concentration was 9694.95 mg / m 3 , which was significantly lower than that of the blank group. 4 The concentration of chlorinated paraffin showed a fluctuating upward trend, reaching a peak of 80422.49 mg / m at the 72nd hour. 3 , CH is produced in the first cycle 4 The average concentration was 37728.7 mg / m 3 , which was lower than that of the blank group. The experimental group treated with tropical Candida also showed a fluctuating upward trend in the first cycle and reached a peak at 96 h, which was delayed compared with the blank group. CH production in the first cycle 4 The average concentration was 49562.05 mg / m 3 , slightly lower than the blank group. The experimental group with addition of brewer's yeast reached a peak of 34836.47 mg / m at 84 h. 3 Compared with the blank group, the peak time was delayed and the peak concentration was significantly lower than that of the blank group. 4The average concentration was 10805.88 mg / m 3 , which had a significant inhibitory effect compared with the blank group.

[0050] The CH production of the blank group in the second cycle (108-192 h), the third cycle (204-288 h) and the fourth cycle (300-384 h) was significantly higher than that of the control group. 4 The average concentrations were 127148.41 mg / m 3 、152848.25 mg / m 3 and 94332.64 mg / m 3 The experimental group with Bacillus subtilis produced CH 4 The average concentrations were 79613.62 mg / m 3 47066.45 mg / m 3 and 51907.41mg / m 3 , which were significantly lower than those in the blank group. 4 The average concentrations of the two groups were lower than those of the blank group, which were 101 and 257.6 mg / m 3 、74324.19 mg / m 3 and 41033.63 mg / m 3 , but the time to reach the peak concentration varies in different cycles. 4 The average concentrations were 113131.95 mg / m 3 、93513.07 mg / m 3 and 68204.4 mg / m 3 , which were lower than those of the blank group. 4 The average concentrations were 22785.86 mg / m 3 、21537.8 mg / m 3 and 36270.59 mg / m 3 Compared with the blank group, the inhibitory effect was significant. This experimental result showed that the four strains reduced CH 4 production, but the inhibitory effects were different.

[0051] like Figure 2 As shown in Figure 2, Saccharomyces cerevisiae inhibits CH production in the anaerobic wastewater system. 4 The best effect was achieved, with the highest inhibition rate reaching 85.91% and the average inhibition rate being 77.12%. The inhibition effect was relatively stable in the first three cycles, but the inhibition rate began to decline from the fourth cycle. 4The inhibitory effect of Lactobacillus acidophilus on CH 4 The inhibition rate of tropical Candida albicans showed an upward trend with the extension of time, reaching a maximum of 56.5% in the fourth cycle, and the average inhibition rate was 38.69%. 4 The inhibition rate showed a trend of first increasing and then decreasing, reaching a maximum inhibition rate of 38.82% in the third cycle, and an average inhibition rate of 20.25%.

[0052] Example 3 This embodiment provides a method for controlling H in a sewage pipe network. 2 S and CH 4 The discharged microbial agent is prepared by mixing Bacillus subtilis liquid and Saccharomyces cerevisiae liquid in a volume ratio of 2:1, and the preparation method thereof is as follows: After activation, Bacillus subtilis was inoculated in LB liquid medium and cultured at 150 r / min at 37°C for 12 h. The culture medium was then removed by centrifugation at 5000 rpm for 10 min. The pure culture of Bacillus subtilis was diluted to OD 0.01 with sterile PBS solution. 600 is 0.3, and the bacterial solution concentration is about 10 8 CFU / mL. After activation, Saccharomyces cerevisiae was inoculated into YPD liquid medium and cultured at 150 r / min at 37°C for 48 h. The medium was then removed by centrifugation at 5000 rpm for 10 min. The pure culture of Bacillus subtilis was diluted to OD using 0.01 mol / L sterile PBS solution. 600 is 1.1, and the bacterial concentration is about 10 8 CFU / mL.

[0053] The prepared pure cultures of Bacillus subtilis and Saccharomyces cerevisiae were compounded in a volume ratio of 2:1, and the prepared bacterial solution was used for subsequent experiments.

[0054] Example 4 This embodiment provides a method for controlling H in a sewage pipe network. 2 S and CH 4 The discharged bacterial agent is prepared by mixing Bacillus subtilis liquid and Saccharomyces cerevisiae liquid in a volume ratio of 1:1, and the preparation method thereof is as follows: After activation, Bacillus subtilis was inoculated in LB liquid medium and cultured at 150 rpm at 37°C for 12 h. The culture medium was then removed by centrifugation at 5000 rpm for 10 min. The pure culture of Bacillus subtilis was diluted to OD 0.01 with sterile PBS solution. 600is 0.3, and the bacterial solution concentration is about 10 8 CFU / mL. After activation, Saccharomyces cerevisiae was inoculated into YPD liquid medium and cultured at 150 r / min at 37°C for 48 h. The medium was then removed by centrifugation at 5000 rpm for 10 min. The pure culture of Bacillus subtilis was diluted to OD using 0.01 mol / L sterile PBS solution. 600 is 1.1, and the bacterial concentration is about 10 8 CFU / mL.

[0055] The prepared pure cultures of Bacillus subtilis and Saccharomyces cerevisiae were compounded in a volume ratio of 1:1, and the prepared bacterial solution was used for subsequent experiments.

[0056] Example 5 This embodiment provides a method for controlling H in a sewage pipe network. 2 S and CH 4 The discharged microbial agent is prepared by mixing Bacillus subtilis liquid and Saccharomyces cerevisiae liquid in a volume ratio of 1:2, and the preparation method thereof is as follows: After activation, Bacillus subtilis was inoculated in LB liquid medium and cultured at 150 rpm at 37°C for 12 h. The culture medium was then removed by centrifugation at 5000 rpm for 10 min. The pure culture of Bacillus subtilis was diluted to OD 0.01 with sterile PBS solution. 600 is 0.3, and the bacterial solution concentration is about 10 8 CFU / mL. After activation, Saccharomyces cerevisiae was inoculated into YPD liquid medium and cultured at 150 r / min at 37°C for 12 h. The medium was then removed by centrifugation at 5000 rpm for 10 min. The pure culture of Bacillus subtilis was diluted to OD using 0.01 mol / L sterile PBS solution. 600 is 1.1, and the bacterial concentration is about 10 8 CFU / mL.

[0057] The prepared pure cultures of Bacillus subtilis and Saccharomyces cerevisiae were compounded in a volume ratio of 1:2, and the prepared bacterial solution was used for subsequent experiments.

[0058] In the above examples 3-5, the obtained liquid bacterial agent can be used directly, and the inoculation amount is 10 7 CFU / mL; it is also centrifuged to make its water content 50%-70% to prepare solid bacterial agent, and the dosage of solid bacterial agent can be controlled at 0.4-0.5kg / m 3 .

[0059] Figure 4The effects of Examples 1, 2, and 5 on the concentrations of TOC (a) and VFAs (b) in sewage are shown in Table 2. Figure 4 As shown in (a), after anaerobic fermentation, the TOC content in the experimental group was significantly higher than that in the blank group. Further analysis of the VFAs content revealed that the concentration of VFAs in the blank group was 0.372 mmol / L, and was mainly composed of acetic acid and propionic acid; while the VFAs concentration in the experimental group with the addition of Bacillus subtilis was 0.374 mmol / L, which was close to that of the blank group, but was mainly composed of propionic acid and valeric acid. The average concentration of VFAs in the Saccharomyces cerevisiae experimental group was 0.513 mmol / L, which was higher than that of the blank group, and was mainly composed of fatty acids with longer carbon chains, such as propionic acid, butyric acid and valeric acid, which may be the reason for the increase in its TOC content, but due to incomplete degradation of organic matter, a large amount of intermediates accumulated. Figure 4 As shown in (b), the concentration of VFAs in the experimental group with the addition of composite bacteria was 1.748 mmol / L, significantly higher than that in the blank group, and propionic acid, butyric acid and valeric acid accounted for 97.31% of the total VFAs content. These fatty acids with longer carbon chains need to be further decomposed and fermented before they can be used by sulfate-reducing bacteria and methanogens. This process is slow, resulting in a reduction in available electron donors, thereby inhibiting H 2 S and CH 4 The production of.

[0060] Test Example 2 The experimental setting was consistent with that of Test Example 1. The blank group was added with 27 mL of 0.01 mol / L PBS solution, and the experimental group was added with 27 mL of the composite bacterial solution obtained in Example 3-5. The final inoculation concentration was 10 7 CFU / mL. The flask was sealed and placed in a constant temperature shaker at 25°C and 120 rpm for subsequent experiments. The H in the gas sampling bag was tested every 12 h. 2 S and CH 4 The concentration changes lasted for 4 days.

[0061] As attached Figure 3 As shown in the figure, different compounding ratios have an effect on the production of H in the sewage anaerobic system. 2 S and CH 4 The concentrations of H 2 S and CH 4 The inhibition rates of H 2 S and CH 4 The inhibition rates of H 2 S and CH 4 The inhibition rates were 65.8% and 62.4% respectively, which showed significant inhibition effects on both gases.

[0062] Application Examples Using the bacterial agent formula of Example 5, the 3 Solid bacterial agent is added to the sewage pipe according to the standard, and the H at the ventilation outlet of the sewage pipe is continuously monitored. 2 S and CH 4 Emission concentration. Figure 5 As shown, H 2 The average S discharge concentration remained at a low level of 4.2 mg / m within one week after the addition of the bacterial agent. 3 , the inhibition rate was 76%, CH 4 Not detected.

[0063] In summary, the microbial composite agent of the present invention is simple to use and has a great effect on inhibiting the discharge of H 2 S and CH 4 It has a very good effect and is suitable for actual production.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A bacterial agent for controlling H2S and CH4 emissions in a sewage pipe network, characterized in that: Includes one or both of Bacillus subtilis and Saccharomyces cerevisiae.

2. The bacterial agent for controlling H2S and CH4 emissions in a sewage pipe network according to claim 1, characterized in that: The solution is prepared from Bacillus subtilis liquid and Saccharomyces cerevisiae liquid in a volume ratio of (1-2): (1-3).

3. The bacterial agent for controlling H2S and CH4 emissions in a sewage pipe network according to claim 2, characterized in that: In the Bacillus subtilis liquid, the effective viable count of the Bacillus subtilis is (1-2)×10 8 CFU / mL; in the brewer's yeast liquid, the effective viable count of the brewer's yeast is (1-2)×10 8 CFU / mL.

4. The method for preparing a bacterial agent for controlling H2S and CH4 emissions in a sewage pipe network according to any one of claims 1 to 3, characterized in that: The steps include: The Bacillus subtilis was cultured in LB liquid medium, and then resuspended in a sterile phosphate buffer solution to obtain a Bacillus subtilis bacterial liquid; And / or, culturing the cerevisiae yeast in a YPD liquid medium, and then resuspending in a sterile phosphate buffer solution to obtain a cerevisiae yeast liquid.

5. The preparation method according to claim 4, characterized in that: The method further comprises the following step: compounding the obtained Bacillus subtilis bacterial solution and the obtained Saccharomyces cerevisiae bacterial solution to obtain the bacterial agent.

6. The preparation method according to claim 4, characterized in that: The LB liquid culture medium uses water as a solvent, and includes 5-15 g / L of peptone, 3-8 g / L of yeast powder, and 5-15 g / L of sodium chloride; the pH value of the LB liquid culture medium is 7.0-7.5, and is sterilized at high temperature at 120-125° C. for 10-30 min; The YPD liquid culture medium uses water as a solvent and includes 10-30 g / L of peptone, 5-15 g / L of yeast extract powder, and 10-30 g / L of glucose; the pH value of the YPD liquid culture medium is 6.0-7.0, and the medium is sterilized at high temperature at 120-125° C. for 10-30 min.

7. The preparation method according to claim 4, characterized in that: The concentration of the sterile phosphate buffer solution is 0.005-0.015 mol / L.

8. The preparation method according to claim 4, characterized in that: Bacillus subtilis was cultured in LB liquid medium at 100-200 r / min at 35-40°C for 12-24 h, then centrifuged at 4000-6000 rpm for 5-15 minutes to remove the medium, and then diluted to OD with sterile phosphate buffer solution. 600 is 0.2-0.4, and a Bacillus subtilis liquid is obtained; Saccharomyces cerevisiae was cultured in YPD liquid medium at 100-200 r / min at 35-40°C for 12-24 h, then centrifuged at 4000-6000 rpm for 5-15 min to remove the medium, and then diluted to OD 0. 600 is 1-1.5 to obtain brewer's yeast liquid.

9. Use of the bacterial agent for controlling H2S and CH4 emissions in a sewage pipe network as described in any one of claims 1 in a method for controlling H2S and CH4 emissions in a sewage pipe network.

10. The use according to claim 9, characterized in that: The bacterial agent is directly added to the sewage for use, and the inoculation amount of the bacterial agent is (1-2)×10 7 CFU / mL; or, the bacterial agent is centrifuged to make its water content 50%-70%, and then prepared into a solid bacterial agent and added to the sewage for use; preferably, the dosage of the solid bacterial agent is 0.4-0.5kg / m 3 .

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